U.S. patent number 7,312,068 [Application Number 10/456,943] was granted by the patent office on 2007-12-25 for capillary pins for high-efficiency microarray printing device.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Donna G. Albertson, Nils W. Brown, Steven M. Clark, Joe W. Gray, Greg Hamilton, John Hanson, Daniel Pinkel.
United States Patent |
7,312,068 |
Pinkel , et al. |
December 25, 2007 |
Capillary pins for high-efficiency microarray printing device
Abstract
This invention provides improved components (e.g. array "pins",
print head, substrate platen, print head platen, and the like) for
microarray printing devices as well as microarray printing devices
incorporating such components. In one embodiment, this invention
provides a microarray print head comprising a plurality of glass or
quartz spotting capillaries disposed in a support that maintains a
fixed spacing between the spotting capillaries and that permits the
spotting capillaries to move in a direction parallel to the long
axis of the capillaries.
Inventors: |
Pinkel; Daniel (Walnut Creek,
CA), Albertson; Donna G. (Lafayette, CA), Gray; Joe
W. (San Francisco, CA), Hamilton; Greg (San Francisco,
CA), Brown; Nils W. (San Francisco, CA), Clark; Steven
M. (Haverton, PA), Hanson; John (Mountain View, CA) |
Assignee: |
The Regents of the University of
California (Oakland, CA)
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Family
ID: |
25403605 |
Appl.
No.: |
10/456,943 |
Filed: |
June 6, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040071603 A1 |
Apr 15, 2004 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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09894863 |
Jun 27, 2001 |
6855538 |
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Current U.S.
Class: |
435/287.2;
422/504; 435/6.11; 436/94; 536/23.1; 536/24.3 |
Current CPC
Class: |
B41J
2/005 (20130101); B41J 2/25 (20130101); C12Q
1/6837 (20130101); C12Q 1/6837 (20130101); C12Q
2565/507 (20130101); Y10T 436/2575 (20150115); Y10T
436/143333 (20150115); Y10T 436/119163 (20150115) |
Current International
Class: |
C12M
3/00 (20060101); C07H 21/04 (20060101); C12Q
1/68 (20060101); G01N 33/00 (20060101) |
Field of
Search: |
;435/6,19.1,183,287.1,287.2 ;422/63,98,100 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1101616 |
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May 2001 |
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EP |
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WO 95/25116 |
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Sep 1995 |
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WO |
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WO 01/71035 |
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Sep 2001 |
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WO |
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Other References
International Search Report. cited by other.
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Primary Examiner: Sisson; Bradley L.
Attorney, Agent or Firm: Beyer Weaver LLP Hunter; Tom
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. Ser. No. 09/894,863,
filed on Jun. 27, 2001 now U.S. Pat. No. 6,855,538, which is
incorporated herein by reference in its entirety for all purposes.
Claims
What is claimed is:
1. A microarray printing device comprising: a microarray printhead,
said microarray printhead comprising a spotting microcapillary,
wherein said microcapillary: comprises a tapered tip has an
internal diameter ranging from about 20 .mu.m to about 100 .mu.m;
has an aperture ID or OD ranging from about 20 .mu.m to about 75
.mu.m; has an outside diameter ranging from about 0.4 mm to about 1
mm; has a load volume ranging from about 0.05 .mu.L to about 1
.mu.L; and wherein said microcapillary is in fluid communication
with a manifold.
2. The microarray printing device of claim 1, wherein said tapered
tip is ground.
3. The microarray printing device of claim 1, wherein said spotting
capillary is fabricated by heating and pulling a quartz or glass
microcapillary tube and then beveling the tip using a glass
grinder.
4. The microarray printing device of claim 1, wherein said spotting
capillary has maximum load volume of about 0.5 .mu.L.
5. The microarray printing device of claim 1, wherein said spotting
capillary has a minimum load volume of about 0.05 .mu.L.
6. The microarray printing device of claim 1, wherein said spotting
capillary has a load volume of about 0.2 .mu.L.
7. The microarray printing device of claim 1, wherein said spotting
capillary is fabricated from a material selected from the group
consisting of glass, a mineral, ceramic, and porcelain.
8. The microarray printing device of claim 7, wherein said spotting
capillary is fabricated from quartz.
9. The microarray printing device of claim 1, wherein said spotting
capillary is fabricated from glass.
10. The microarray printing device of claim 1, wherein said
spotting capillary has an inside diameter (bore width) at the tip
of less than about 100 .mu.m.
11. The microarray printing device of claim 1, wherein said
spotting capillary has an inside diameter (bore width) at the tip
of less than about 50 .mu.m.
12. The microarray printing device of claim 1, wherein said tapered
tip is beveled.
Description
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[Not Applicable]
FIELD OF THE INVENTION
This invention pertains to the field of high-density microarray
production. In particular, this invention provides methods and
devices that permit high-density arrays to be printed with
significantly smaller feature size and spacing and greatly improved
reagent usage.
BACKGROUND OF THE INVENTION
The immobilization of test molecules or "probes" on array supports
has had a significant impact on drug discovery, medical diagnostic
methods, and basic research. The use of high-density microarrays of
organic molecules permits literally thousands of assays to be
simultaneously performed on one or more samples. Using high-density
microarrays, numerous analytes can be simultaneously detected
and/or quantified permitting the rapid characterization of complex
systems (e.g. complex assays for gene expression). High-density
microarrays are also useful for "high-throughput" screening assays,
diagnostics, and in many other contexts. The ability to manufacture
microarrays in an efficient and cost-effective manner is of
considerable interest to researchers worldwide and of significant
commercial value.
In general, microarrays of greater density are preferred. A higher
density array typically allows more assays to be performed
simultaneously and/or, for lower sample volumes to be used for the
same number of assays. In providing large, high-density arrays of
molecules (e.g., probes or analytes) there are a number of
considerations. The array elements (e.g. dots) should be
substantially reproducible in size, particularly if one wishes to
quantify an analyte. In addition, the array elements should be
consistently and reliably positioned, and should be highly
reproducible.
The basic approaches for generating arrays of test molecules such
as nucleic acid, protein or other organic molecules fall into two
general categories. In the first such approach the test molecules
are directly synthesized onto the array support, while in the
second such approach the test molecules are attached to the support
post-synthetically. Each approach has its own limitations and
drawbacks. For example, when an array is created by direct
synthesis onto an array support, the efficiency of each synthetic
step affects the quality and integrity of molecules forming the
array. The magnitude of the problem increases with the complexity
of the individual molecules, potentially resulting in an
undesirable percentage of incorrectly synthesized molecules and
incomplete sequences. Such contaminants can interfere with
subsequent use of the array.
In addition, synthetic approaches (e.g. as described by Southern et
al. (U.S. Pat. Nos. 5,770,367, 5,700,637, and 5,436,327), Pirrung
et al. (U.S. Pat. No. 5,143,854), Fodor et al. (U.S. Pat. Nos.
5,744,305 and 5,800,992), and Winkler et al. (U.S. Pat. No.
5,384,261), are generally unable to construct microarrays of large
macromolecules. Such technologies can also be expensive and
difficult to implement.
In contrast, the second approach to array production allows the
desired molecules to be produced (e.g. synthesized, isolated,
amplified, e.g.) by conventional methods prior to their formation
into an array. Consequently, the quality of the arrayed molecules,
and thus the quality of the resultant array, is potentially greater
than that produced by the direct synthesis approach.
Such "spotting" approaches include, but are not limited to inkjet,
and direct surface contact printing. Inkjet devices require high
reagent volumes and risk "probe" degradation during
volatilization.
Direct surface contact printing (see, e.g., U.S. Pat. Nos.
4,981,783, 5,525,464, 5,770,151, and 5,807,522), are limited in
their ability to reliably, reproducibly, and uniformly apply the
array elements to the array substrate. Reagent usage is also
relatively inefficient, and array density is limited.
SUMMARY OF THE INVENTION
The present invention provides improved components (e.g. array
"pins", print head, substrate platen, print head platen, and the
like) for microarray printing devices as well as microarray
printing devices incorporating such components. In particular,
methods and devices of this invention permit high-density arrays to
be printed with significantly smaller feature size and spacing, and
greatly improved-reagent usage.
In one embodiment this invention provides a microarray print head,
said print head comprising a plurality of glass or quartz (or other
mineral), or ceramic, or porcelain, or ceramic spotting capillaries
disposed in a support that maintains a fixed spacing between the
spotting capillaries and that permits the spotting capillaries to
move in a direction parallel to the long axis of the capillaries
(i.e. the spotting capillaries can slide through the support).
Preferred spotting capillaries are microcapillary tubes and
particularly preferred spotting capillaries have a tapered tip
(e.g. a ground, beveled tip). The capillaries can have any
desirable cross-section (e.g. round, ovoid, square, triangular,
irregular), however preferred capillaries are round in
cross-section.
In certain preferred embodiments, the capillaries have a maximum
load volume of about 0.5 mL. In certain preferred embodiments, the
spotting capillaries have a load volume of about 0.2 mL.
Preferred print heads comprise at least 4 spotting capillaries,
preferably least , 4, 16, 32, 64, or 128 spotting capillaries and
in certain preferred embodiments, the spacing between two adjacent
spotting capillaries is about 3 mm or less, center to center.
In certain preferred embodiments, the spotting capillaries have
detents where the spotting capillaries have a rest position in
which the detents contact a support stopping the movement of the
spotting capillaries in a direction toward the substrate that is to
be printed. The print head can also comprise a spring attached to a
spotting capillary where, in the absence of a force against the
printing tip of the spotting capillary the spring returns said
spotting capillary to a rest position. The print head can be
provided separately or can be found as a component in a microarray
printing device.
In preferred embodiments, the spotting capillaries are in fluid
communication (e.g. via flexible capillary tubing) with a manifold.
In preferred embodiments, the manifold comprises a common port and
a plurality of individual ports where an aperture into an
individual port is disposed inward of the inside wall of the
manifold. The manifold can be connected to a gas and/or vacuum
source.
In another embodiment this invention provides a platen for
positioning a substrate holder or a print head in a microarray
printing device. A preferred platen comprises a support surface
attached to a single guide rail such that the support surface can
move along the guide rail, and motion of the support is constrained
in a direction normal to the guide rail, and a flexible coupling to
an actuator wherein the flexible coupling is rigid or stiff in a
direction parallel to the guide rail, but is flexible in another
direction. The platen also, optionally, comprises an encoder (e.g.
optical encoder, magnetic encoder, electronic encoder, etc.) that
encodes the position of said platen along said guide rail. In
certain embodiments, the platen is attached to the rail by two
bearings. Preferred flexible couplings include, but are not limited
to a flexible sheet coupling (e.g. sheet metal, sheet plastic,
etc.), a rod bearing, a ball bearing, a pin bearing and the like.
Preferred actuators include, but are not limited to a stepping
motor, a linear motor, a lead screw, and the like. In certain
embodiments, the platen can further comprise a holder (e.g. a slide
holder) for one or more microarray substrates. In certain
embodiments, the platen is attached to a microarray print head
(e.g. directly or through a movable stage). Preferred print heads
in such cases include, but are not limited to any of the print
heads described herein.
In still another embodiment, this invention provides a microarray
printing device comprising a microarray print head (e.g. as
described herein); and a microarray substrate holder attached to a
platen (e.g. a platen as described herein). Preferred microarray
printers can print at least 2,000, more preferably at least 5,000
array elements per spotting capillary per load. Preferred
microarray printers can print array elements with a precision of at
least 30 .mu.m and/or with an average inter-element spacing of 130
.mu.m or less. Preferred microarray printers can print 200 or more
microarray substrates in a run. Particularly preferred microarray
printers of this invention utilize pressure and/or vacuum to
control reagent loading or dispensing. Certain microarray printers
comprise the spotting capillaries are in fluid communication with a
manifold. A preferred manifold comprises a common port and
individual ports where an aperture into an individual port is
disposed inward of the inside wall of the manifold. In certain
preferred embodiments, the microarray printing device can loads
reagents from a microtiter plate comprising at least about 864
wells.
This invention also provides a method of printing a microarray
(e.g., a nucleic acid and/or protein and/or small organic molecule
microarray). The methods involve providing an array substrate in a
microarray printing device comprising one or more of the elements
(e.g. spotting capillaries, print head, array substrate platen,
print head platen, and the like) as described herein, providing a
series of solutions comprising the reagents that will form features
of the microarray; and operating said microarray printing device to
print the microarray. In preferred methods the microarray printing
device prints a microarray comprising at least 1,000 different
array elements. In preferred methods the microarray printing device
prints a microarray comprising having an average inter-feature
spacing of about 130 .mu.m or less. Preferred array substrates
include, glass, quartz or other minerals, metals, ceramics,
plastics, metal coated glass, metal coated plastic and the like. In
preferred methods the microarray printing device applies negative
pressure to load a spotting capillary and/or positive pressure to
dispense from a spotting capillary. In preferred embodiments, the
method involves loading feature-forming reagents from a microtiter
plate comprising at least about 864 wells.
In still another embodiment, this invention provides (printed)
microarrays. Preferred printed microarrays comprise at least about
1000 different array elements on an array substrate, where the
array elements are separated by an average center to center spacing
of about 130 .mu.m or less, preferably about 100 .mu.m or less,
more preferably about 80 .mu.m or less. Where the arrays are
nucleic acid and/or protein arrays, the protein or said nucleic
acid is preferably not a chemically synthesized protein or nucleic
acid. Particularly preferred microarrays include nucleic acid
nucleic acid microarrays. In certain embodiments, the nucleic acids
comprising such microarrays have an average length greater than
about 50, preferably greater than about 100, 200, or 500
nucleotides, more preferably greater than about 1000 nucleotides.
The molecules comprising the array features are preferably adsorbed
to the array substrate. In certain nucleic acid or protein arrays
the nucleic acid or protein is not covalently coupled to the array
substrate (e.g. not coupled directly or though a linker to a
terminal nucleotide or amino acid). In particularly preferred
microarrays, the features comprising the arrays are at an average
density of about 40,000/cm.sup.2 or greater.
DEFINITIONS
The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers. The term also includes
variants on the traditional peptide linkage joining the amino acids
making up the polypeptide.
The terms "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein refer to at least two nucleotides covalently
linked together. A nucleic acid of the present invention is
preferably single-stranded or double stranded and will generally
contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et al.
(1993) Tetrahedron 49(10): 1925) and references therein; Letsinger
(1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J.
Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acids Res. 14:
3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988)
J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) Chemica
Scripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic
Acids Res. 19:1437; and U.S. Pat. No. 5,644,048),
phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111 :2321,
O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides
and Analogues: A Practical Approach, Oxford University Press), and
peptide nucleic acid backbones and linkages (see Egholm (1992) J.
Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl.
31: 1008; Nielsen (1993) Nature, 365: 566; Carlsson et al. (1996)
Nature 380: 207). Other analog nucleic acids include those with
positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA
92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684,
5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl. Ed.
English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc.
110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide
13:1597; Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate
Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan
Cook; Mesmaeker et al. (1994), Bioorganic & Medicinal Chem.
Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17;
Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones,
including those described in U.S. Pat. Nos. 5,235,033 and
5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
Carbohydrate Modifications in Antisense Research, Ed. Y. S. Sanghui
and P. Dan Cook. Nucleic acids containing one or more carbocyclic
sugars are also included within the definition of nucleic acids
(see Jenkins et al. (1995), Chem. Soc. Rev. pp169-176). Several
nucleic acid analogs are described in Rawls, C & E News Jun. 2,
1997 page 35. These modifications of the ribose-phosphate backbone
may be done to facilitate the addition of additional moieties such
as labels, or to increase the stability and half-life of such
molecules in physiological environments.
The terms "spotting capillary" and "pin" or "printing pin" are used
synonymously to refer to the structure that is used to contact a
microarray substrate and thereby deposit a reagent to form a
microarray feature on that substrate. Unlike many printing pins,
however, the spotting capillary is typically a tube and, while not
limited to such, in certain preferred embodiments, display a round
cross section.
An "array substrate" refers to the surface or support on which a
microarray is printed. Array substrates include, but are not
limited to glass, quartz or other minerals, ceramic, porcelain,
metal, and metal-coated glass.
An "array feature" or "array spot" refers to a reagent or reagents
deposited at a location on an array surface. Typically a feature is
characterized by the presence of one or more specific molecules
(e.g. particular proteins, nucleic acids, etc.).
A "guide rail" refers to a rail or other device that directs or
orients the movement of a platen as described herein. In certain
embodiments, guide rail can take any of a number of forms
including, but not limited to T-shaped, round, triangular, square,
ovoid, and the like. The guide rail is typically coupled to the
platen through one or more bearings that permit motion of the
platen in one direction (along one axis), but restrict motion in
other directions. In certain preferred embodiments, the guide rail
is configured to to control the motion each degree of freedom of
the spotter (e.g. the platten and/or print head) the parts of the
robot with the minimal number of restraints that are required by
Euclidian geometry, e.g. as described herein.
A "print cycle" refers to the sequence of events involved in
printing an array feature.
The term "microarray" refers to an array comprising at least about
10, preferably at least about 50, more preferably at least about
100, still more preferably at least about 500 or 1000, and most
preferably at least about 10,000, 40,000, 100,000, or 1,000,000
different and distinct features. Preferred microarrays have an
average feature density greater than about 100/cm.sup.2, more
preferably greater than about 1000/cm.sup.2, still more preferably
greater than about 5,000/cm.sup.2, even still more preferably
greater than about 10,000/cm.sup.2, and most preferably greater
than about 20,000/cm.sup.2, 40,000/cm.sup.2, 60,000/cm.sup.2, or
even 80,000 cm.sup.2.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B illustrates a microarray printer print head 10 of
this invention. FIG. 1A illustrates a print head 10 comprising a
series of guide plates 14 that support and position the spotting
capillaries 12. The downward motion of the spotting capillaries is
limited by a detent 20 and the spotting capillaries are returned to
the "extended" position by a spring 18 compressed between the
detent and a spring capture plate 22. The spotting capillaries
communicate to a manifold through a flexible capillary tubing 24.
FIG. 1B illustrates a print head capable of mounting 64 spotting
capillaries on 3 mm centers for 864 well microtiter plates. In this
illustration, 16 spotting capillaries are in use.
FIG. 2 illustrates a glass or quartz spotting capillary of this
invention showing the inside diameter 32, the outside diameter 34,
and the bevel 30 at the tip.
FIG. 3 illustrates a two-rail positioning platen compared to a
preferred one-rail platen.
FIG. 4 illustrates one embodiment of a platen used to position an
array substrate in an array printing device.
FIG. 5 illustrates an exploded view of a platen used to position an
array substrate in an array printing device.
FIG. 6 illustrates one embodiment of a platen used to position a
microarray print head in an array printing device.
FIG. 7 schematically illustrates vacuum and pressure plumbing of a
microarray printer of this invention.
FIG. 8 illustrates a preferred manifold design.
FIG. 9 illustrates the print head and its associated plumbing.
FIG. 10 illustrates unidirectional flow through a manifold
according to the methods and devices of this invention. The
manifold 64 is connected to flexible tubing 84a leading to a vacuum
source and to flexible tubing 84b leading to a pressure source.
Flow is unidirectional in the direction of the open arrow.
FIG. 11 schematically illustrates controls for the pressure side of
the pressure control system for printhead operation.
FIG. 12 schematically illustrates controls for the vacuum side of
the pressure control system for printhead operation.
FIG. 13 illustrates a scheme for passive magnetic control of
printing pin/capillary position.
FIG. 14 illustrates a scheme for active magnetic control of
printing pin/capillary position.
DETAILED DESCRIPTION
This invention pertains to a microarray printer that can be used to
manufacture microarrays (e.g. of biochemical samples) by direct
contact printing. The array printer of this invention is capable of
printing microarrays at higher feature density, with greater speed
and lower cost than previous microarray printing devices.
Without being bound to a particular theory, these efficiencies are
achieved by the use of a combination of novel features. A novel
printing pin permits vastly more efficient reagent usage and a
greater number features to be printed per load. This reduces
reagent costs, and because the pin does not require repeated
refills during the printing process, the printing operation
proceeds more rapidly.
In addition, positive control of reagent flow using pressure and
vacuum, also improves reagent capture and delivery and reduces the
incidents of mis-prints due to pin blockage during loads or print
steps. In addition, positive pressure control keeps the liquid at
the tip of the printing capillary. This significantly improves
printing reliability.
A novel design for the substrate support permits the use of a
larger positioning substrate that can hold a greater number of
array substrates (e.g. more than 10, preferably more than 20 or 50,
still more preferably greater than 100, and most preferably greater
than 150, 200, 250, 300, or even greater than about 500 standard
slide-sized substrates) and position each array with greater
accuracy and precision. The substrate support is kinematically
constrained so that printing substrates are more reproducibly
positioned even at rapid accelerations and decelerations, thereby
permitting a print run to proceed with greater rapidity, i.e.,
decreasing effective print time and reducing printing costs, and to
print arrays with a higher density of spots.
A related kinematic support design for the print head permit rapid
acceleration and deceleration of the print head and reproducible
positioning. The rapid precise positioning of the array substrate
combined with the rapid positioning of the print head again,
significantly reduces the time required for a print cycle thereby
reducing costs, and increases the utility of the arrays by allowing
production of a higher density of spots.
These features combine to make possible the efficient printing of
microarrays at extremely high efficiency with low reagent usage,
and previously unobtainable feature spacing for printed
microarrays.
I. Printing Pins and the Print Head
In one embodiment, this invention provides for print head 10 for
printing microarrays and a microarray printer comprising such a
print head. As illustrated in FIG. 1, in one preferred embodiment,
the print head 10 comprises a plurality of spotting capillaries 12
disposed in a support 14 that maintains a fixed spacing between the
spotting capillaries and that permits the spotting capillaries to
move in a direction parallel to the long axis of said capillaries
(i.e., the spotting capillaries can slide in the support).
The spotting capillaries 12 are preferably cylinders (e.g.
capillary tubes) made of a rigid material such as glass, quartz or
other mineral, ceramic, brittle plastic (e.g. acrylic), and the
like. It was a surprising discovery of this invention that
glass-like materials such as glass or quartz or ceramic could be
effectively used as spotting capillaries. Moreover, particularly
when fabricated and utilized as described herein, such glass,
quartz or ceramic spotting capillaries have a useful lifetime
vastly greater than that observed for the commonly utilized metal
pins. Indeed, we have yet to determine the maximum lifetime of the
spotting capillaries described herein, while it is believed that
metal spotting are quite limited in their useful life.
The spotting capillaries used in this invention can be of any
convenient size, however, in preferred embodiments (see, e.g., FIG.
2), the spotting capillaries are microcapillaries, with an inside
diameter (bore width) at the tip of less than about 100 .mu.m,
preferably less than about 75 .mu.m, more preferably less than
about 50 .mu.m, and most preferably less than about 30 .mu.m, 25
.mu.m or even less than about 20 .mu.m. The lower size limit (bore
diameter) can be determined by practical considerations of plugging
given the occurrence of particulate matter in some preparations.
Thus, there can be practical size limits on the printing capillary
tip opening size given the care (cleanliness) with which the
printing solutions are prepared. If the inside diameter of the
capillary is too small and the capillary is too long, then the flow
resistances of the capillary can impede the ability to rapidly draw
cleaning solutions etc. through it. Thus, in certain preferred
embodiments, the capillaries are used that have a larger ID away
from the tip, and the capillary diameter (ID and OD) is reduced at
the tip to produce a small feature (spot) size. The load volume
(loaded fluid volume) of the spotting capillary is typically 1
.mu.L or less, preferably 0.5 .mu.L or less, more preferably 0.25
.mu.L or less, and most preferably 0.2 .mu.L or less or even 0.1
.mu.L or less.
As indicated above, the spotting capillaries need not have a
constant internal diameter. The diameter (ID and especially OD) at
the aperture (spotting face) of the spotting capillary will, in
part, determine the minimum feature size of the spotted microarray.
Thus, smaller aperture and external tip diameters are preferred.
Particularly preferred aperture diameters (ID or OD) are less than
about 75 .mu.m, more preferably less than about 50 .mu.m, and most
preferably less than about 30 .mu.m, 25 .mu.m or even less than
about 20 .mu.m or 15 .mu.m. In certain embodiments, the diameter of
the internal channel expands to a maximum of about 100 .mu.m,
preferably about 75 .mu.m, more preferably about 50 .mu.m. In one
embodiment, illustrated in FIG. 2, the spotting capillary has an
aperture diameter of about 30 .mu.m or less and a maximum internal
diameter of about 75 .mu.m or less. The capillary holds about 0.2
.mu.L or less, preferably about 0.1 .mu.L or less.
The spotting capillary outer diameter determines the minimum
inter-pin (inter-capillary) spacing, and the inter-pin spacing
determines the minimum spacing of the reservoir(s) from which the
print head can load reagents. Preferred spotting pins have an
outside diameter of about 1 mm or less, more preferably of about
0.7 mm or less, and most preferably of about 0.4 mm or less. The
0.4 mm spotting capillaries can be mounted very close together and,
with such close spacing, the print head can load reagents from
standard 96 well, 384, well, 864 well, and 1536 well microtiter
plates. In preferred embodiments, the center to center spacing of
the spotting capillaries is about 10 mm or less, preferably about 5
mm or less, more preferably about 3 mm or less, and most preferably
about 2 mm or even 1 mm or less. Such close spotting capillary
spacing can be achieved, e.g. using the print head designs
illustrated herein, allowing the use of even higher density sample
reservoirs.
The support(s) for the spotting capillaries can take any of a
number of a number of forms. For example, in one embodiment, the
support can comprise a number of channels drilled, etched, or cast
in a single metal or plastic piece. The channels then act as guides
for the spotting capillaries. Alternatively, the support can be
fabricated as by joining a collection of tubes e.g. metal tubes.
The tubes can be glued or welded together to form a single support
structure, each of the tubes acting as a channel for housing a
spotting capillary.
In a particularly preferred embodiment, as illustrated in FIG. 1A,
the spotting capillaries are supported and positioned by a series
of guide plates 14, and optionally, by a guide cylinder 16. To
prevent breakage of the spotting capillaries e.g., do to the
repetitive contacting with the printing substrate, the spotting
capillaries are capable of sliding through the guide plates, e.g.
when they contact the spotting substrate. The spotting capillaries
are then returned to their "extended" position by a spring 18.
While FIG. 1A is illustrated with a coiled spring, it will be
appreciated that any of a variety of springs can be used. These
include, but are not limited to deformable elastic masses,
deformable elastic membranes, "rubber bands", air pressure, and the
like.
The extended position of the spotting capillaries is limited by a
detent 20. The detent can take any convenient form. For example,
the detent could comprise a set screw (preferably plastic so as not
to damage the spotting capillary), a drop of epoxy or other resin,
and the like. In one particularly preferred embodiment, the detent
is a disk attached to the spotting capillary. In one embodiment the
detent stops up against the pin guide plate The "downward" extent
of the spotting capillary can be determined by the position of
attachment of the detent to the spotting capillary. Alternatively,
the guide plate can further comprise an adjustment means (e.g. a
set screw, a shim, etc.) for each spotting capillary that can be
used to adjust the downward travel for each spotting capillary.
In certain embodiments, the spring typically rests against a
resisting surface, e.g. a spring capture plate 22.
It was also a discovery of this invention that spotting
capillaries, particularly when fabricated of glass, quartz, other
minerals, or ceramic or porcelain, show a dramatically improved
lifetime, when the outer edges of the spotting tip of the spotting
capillary, are not flush with the spotting face. Thus, in preferred
embodiments, the outer edge of the spotting tip is beveled (see,
e.g., 30 in FIG. 2).
The print head typically comprises a plurality of spotting
capillaries. Preferred print heads comprise at least two spotting
capillaries, more preferably at least 4 spotting capillaries, still
more preferably at least 8 or at least 16 spotting capillaries, and
most preferably at least 32, 64, 128, or 256 spotting capillaries.
Depending on the application, a print head can be configured to use
fewer than the maximum number of available spotting capillaries.
FIG. 1B illustrates a print head capable of mounting 64 pins on 3
mm centers for 864 well microtiter plates. In this instance, 16
pins are in use.
The print heads of this invention can be fabricated using standard
machining and glass handling techniques well known to those of
skill in the art. The spotting capillaries are preferably
fabricated by casting or by pulling a quartz or glass
microcapillary tube using a commercially available microcapillary
puller (e.g. Sutter Instrument P-2000 Capillary Puller). In
particularly preferred embodiments, the microcapillary tip is then
beveled using a glass grinder. Such spotting capillaries can be
made to order by commercial production houses.
II. Magnetic Control of Printing Pins/Capillaries
In certain embodiments, the printing capillary position can be
controlled by magnetic methods. Both passive and magnetic control
methods are contemplated by this invention.
A) Passive Magnetic Control of Printing Pins/Capillaries.
A passive magnetic control system is schematically illustrated in
FIG. 13. This figure illustrates a portion of a print head
comprising a printing capillary 12 and guide plates 14a and 14b.
The capillaries have affixed thereto a magnetic material 202.
Similarly, guide plate 14a can be a magnetic material. One or both
of 202 and 14a is magnetic and a "passive" magnetic attraction "M"
pulls the capillary 12 downwards. The downward travel of the
capillary is stopped by optional spacer 200.
The open arrow indicates the direction of motion of the print head
during a printing operation. When the print head is loward, the
printing capillaries contact the array substrate. The are then no
longer held doun by the magnetic force "M". When the print head is
raised, the magnetic attraction between 200 and 14a pulls the
pins/capillaries back to the reference position. This system makes
is easy to change printing pins. They can be changed just by
pulling them out of the top of the print head.
B) Active Magnetic Control of Printing Pins/Capillaries.
An active magnetic control system is schematically illustrated in
FIG. 14. In this embodiment, the printing pins are contacted with
the array substrate using electromagnetic means. The system offers
two advantages. First, the printing pins move, while the print head
remains stationary, so printing speed is increased. Second, the
printing pins can readily be changed, simply by lifting them out of
the top of the print head.
In this embodiment, guide plate 14a is an electromagnet. Each
printing pin/capillary has affixed thereto a material 202 that is
attracted by a magnetic field (e.g. a ferrous material, a magnet,
etc.). A spring or elastic material 204 lifts the pins when the
electromagnet is not activated. When the electromagnet 14a is
activated, the pins are drawn downward to contact the array
substrate thereby depositing array features.
The system is simple to build since only one electromagnet is
required regardless of the number of pins/capillaries.
III. Platens for Array Substrate and/or Print Head Positioning
To reliably print an array at high feature density (spots/cm.sup.2)
it is desirable to reliably and consistently position the spotting
capillaries on the microarray substrate. The more precisely and
consistently the print head can be positioned relative to the
microarray substrate(s), the more possible it becomes to print
arrays at a higher feature density.
Printing a larger number of arrays at a time, however, requires a
larger array substrate support (platen). The larger the platen, the
more difficult it becomes to reliably and consistently position it
relative to print head. One approach to solve this problem is
illustrated in FIG. 3. The platen illustrated on the left utilizes
two guide rails 42 to minimize torque and yaw and hysteresis of the
platen introduced by the actuator which moves the platen, e.g. in
the .+-.Y direction. To accurately print arrays, it is desirable to
accurately position the platen to tolerances better than 50 .mu.m.
Such precise positioning requires extremely good bearing alignment
for the four bearings and the guide rails must be extremely
parallel. The rails and bearings must stay aligned throughout the
printing operation. One of skill in the art will appreciate that
such a device will tend to jam and/or introduce positioning
imprecision as the actuator drives the platen through jammed
positions if the alignment is not adequate. Leaving "play" in the
bearings to allow motion results in positional imprecision. In
still other standard types of arrayers the guide rails, e.g. as
illustrated in the embodiment on the left in FIG. 3 are
incorporated into the actuator. These rails are closely spaced and
permit slight random yaw motions of the platen. For large platens,
even this slight yaw results in a positional uncertainty that
increases as one moves away from the center of the platen and
decreases array element accuracy and therefore array element
density.
Such difficulties are solved with the platens of this invention.
One embodiment of a platen 40 of this invention is illustrated by
the platen on the right in FIG. 3, and in FIG. 4. In certain
embodiments, the plattens of this invention utilizes a single guide
rail 42 to constrain the position of the support surface 44 that
bears the array substrates 56. The guide rail is typically coupled
to the platen through one or more bearings that permit motion of
the platen in one direction (along one axis), but restrict motion
in other directions. In certain preferred embodiments, the guide
rail/platten configuration is designed to control the motion each
degree of freedom of the spotter (e.g. the platten and/or print
head) with the minimal number of restraints that are required by
Euclidian geometry. Thus, for example, two points determine a line,
three points determine a plane etc. To limit degrees of freedom
around the guide rail, two coupling points (e.g. bearings) are used
to constrain the direction of motion (e.g. in a line), and a
coupling point is used at a location off of the line to limit the
degrees of freedom of the platten, a plane. The attachment to this
third point provides some flexibility in certain degrees of freedom
etc. since the guide rail may not be accurately straight. In this
example, it is not desired to place three bearings on the guide
rail since that is more than required and if the rail is slightly
curved the bearings will bind. Similarly one bearing on the rail is
generally insufficient, because that will allow the platten to
rotate.
Illustrative embodiments, of these principles are provided in FIG.
3, and in FIG. 4. As illustrated, the platen utilizes a single
guide rail 42 to constrain the position of the support surface 44
that bears the array substrates 56. The support surface
communicates with a guide rail 42 through two bearings 46, the
minimal number required to define linear motion. The support
surface is coupled to an actuator 48 through a flexible coupling
50.
The bearing(s) 46 and the guide rail 42 prevent the platten (e.g.,
the support surface) from yawing in response to a force created by
the actuator. However, because there is only a single rail, there
are no difficult alignment problems. Straightness of the guide rail
is also not critical because any rail deformation will be constant
and reproducible, i.e., repeated positioning of the array
substrates will be consistent, and the bearings will not bind due
to possible curvature in the rail. An encoder 58 accurately encodes
the location of the platen. The encoder can be external to the
actuator as shown in the drawing, or it can be built into the
actuator as is common in commercially available actuators.
The support surface is coupled to the actuator through a flexible
coupling 50 that is rigid in the direction of travel (.+-.Y
direction in FIG. 3), but compliant in all other directions. This
permits the actuator to accurately position the platen (e.g. in the
Y direction as shown), while not jamming or binding in other
directions.
The distance between the two bearings on the guide rail determines
how much yaw motion will be permitted in the platten. If the
bearings allow a certain lateral motion, that will be translated
into a yaw of the platten. The positioning precision is related to
the length and width of the slide platten in relation to the
distance between the two bearings on the guide rail. It is
desirable to provide very high reproducibility in the positioning
of the plattens, but the absolute accuracy is not so critical. Thus
if the guide rail is curved a bit, the platten will yaw as it
moves, but that motion will be very reproducible so that the next
array spot is printed, it is properly placed relative to the
previous spots, although the entire array on one substrate will be
positioned slightly differently than on a substrate at a different
location on the platen. Thus the system is designed to give
extremely good performance on the relative positions of spots in an
array.
The embodiment illustrated in FIG. 4 allows slight motion in the X
direction, and yaw, roll and pitch compliance. The system is stiff
in the Y direction. One thing to note is that with the flexing,
especially pitch, the locations on the platen will vary compared to
the encoder, when the encoder is on the motor (actuator) carriage.
As long as these variations are reproducible, then the positioning
is reliable.
The flexible coupling 50 illustrated in FIG. 4 is a flexible sheet
(e.g., sheet metal. This flexure gives freedom in yaw, roll and X,
and is stiff in Y, the direction of platen travel. If pitch freedom
is desired, it is possible to introduce additional flexible
couplings 54. Alternatively, a bearing can be used. The two
flexures 54 will be stiff in the Y direction as long as the platen
does not lift up under acceleration, and will be stiff in yaw so
that all of the yaw compliance will be taken care of in the
vertical sheet.
In certain embodiments, a pivot 52 is provided. In various
embodiments, the pivot 52 includes, but is not limited to two
points across the width (into the drawing in the side view), a
cylinder etc. In this design, pitch motion will require the upper
mounting plate to slide across the pivot, so this is preferably
lubricated and/or fabricated of low-friction materials, etc.
FIG. 5 illustrates one embodiment of an array substrate platen in
an exploded view.
FIG. 6 illustrates one embodiment of a platen used to move the
print head in a microarray printer of this invention in a .+-.X
direction (normal to the direction of motion of the array substrate
platen). In this embodiment, the platen (support surface) is
oriented vertically. A microarray print head 10 is attached to the
support surface 44. In the embodiment illustrated in FIG. 5, the
actuator 48 is a motor (e.g. a linear stepping motor). The actuator
48 is coupled to the support surface through a flexible coupling
50. The couplings are disposed such that deflection of the support
surface is constrained (stiff) in the X direction (the direction of
motion), and stiff in the Y direction, but compliant in yaw. The
coupling 50a flexes to relieve Z, yaw, and roll of the platten. The
platen rides along a truss 58 that bears a guide rail 42. In a
preferred embodiment, the support surface 44 is trapezoidal in
shape with the wide side of the trapezoid disposed along the guide
rail. This permits the rail bearings to be widely separated and
thereby minimize rotation of the support surface. Use of the
trapezoidal shape minimizes support surface mass permitting more
rapid acceleration and deceleration. The print head 10 is mounted
on a Z stage 60 that controls vertical movement of the print
head.
Because the moment arm between the flexible motor coupling 50 and
the print head is large compared to the moment arm between the
print head and the guide rail 42, rotations or deflections at the
motor coupling have minimal effect on the position of the print
head.
Using the teachings provided herein, one of skill would recognize
numerous embodiments for the flexible couplings used in the platens
of this invention. In certain embodiments, as indicated above, the
flexible couplings comprise flexible sheets (e.g. sheets of metal,
plastic, or other flexible material). The sheets are selected of
materials that are stiff in tension, but capable of bending in
other directions. Other flexible couplings include, but are not
limited to ball bearings, rod bearings, pin bearings, and the
like.
A wide range of encoders can be used to encode the position of the
platen/support surface. Encoders are well known to those of skill
in the art and include, but are not limited to optical encoders,
mechanical encoders, magnetic encoders, and electronic encoders.
Various electronic encoders include, but are not limited to
encoders that convert the change in resistance of a potentiometer
or the change in capacitance of a capacitor into a movement or
position. Optical encoders include, but are not limited to encoders
that convert an optical signal, e.g. a bar code, an interferometric
measurement, etc. into a movement or position. Similarly, magnetic
encoders include encoders that a change in magnetic flux or field
into a movement or a position. Suitable encoders (e.g. with a
positional accuracy greater than about 50 .mu.m, preferably with a
positional accuracy greater than about 25 .mu.m, more preferably
with a positional accuracy greater than about 10 .mu.m, and most
preferably with a positional accuracy greater than about 5 .mu.m,
greater than about 2 .mu.m, or greater than about 1 .mu.m are
commercially available.
The actuator can be any device or means capable applying a force to
the platens of this invention and driving them in a plus or minus
direction along the guide rail. Suitable actuators include, but are
not limited to stepping motors, linear induction motors, pneumatic
actuators, solenoids, piezo-electric actuators, lead screws, and
the like. Suitable actuators, and associated motion control
products are commercially available from a wide variety of
companies (see, e.g., Biorobotics (U.K), Parker Daedal, Irwin, Pa.,
and the like).
It is noted that in certain preferred embodiments, of the present
invention, the slide support platen, with an encoder precision of
about 2 .mu.m achieves a spot (array element) precision of about 10
to about 20 .mu.m at any array substrate location on the support
surface. Particularly preferred platens achieve a spot (array
element) precision of better than about 5 .mu.m, more preferably
better than about 2 .mu.m, and most preferably better than about 1
.mu.m, or 500 nm, or 200 nm at any array substrate location on the
support surface.
Similarly, the print head positioning platen achieves a precision
of about .+-.10 .mu.m or less, more preferably about .+-.5 .mu.m or
less, and most preferably about .+-.3 .mu.m or less over the entire
slide (array substrate) support surface.
IV. Positive and Negative Pressure Control for Sample Loading and
Dispensing
A) Pressure Control Systems.
In a particularly preferred embodiment, the microarray printing
devices of this invention utilize positive pressure and negative
pressure (vacuum) to control sample loading and dispensing. Each
"active" spotting capillary 12 is in fluid communication, e.g. via
capillary tubing 70 with a manifold 64 (see, e.g., FIGS. 6, 7, 8,
and 9) that permits the application of pressure or vacuum to the
spotting capillaries.
A preferred plumbing scheme is illustrated in FIG. 7. In preferred
embodiments, the gas flow (e.g. air, nitrogen, argon, etc.) flows
through the manifold always in the same direction for all
operations. This assures that any liquid drops that may be left in
the tubing always will be forced to move toward the waste bottle
and will not be blown back into the manifold. If droplets do get
into the manifold they may block the supply of printing pressure to
one or more pins, thus reducing the reliability of the
printing.
In general, the plumbing system comprises a pressure source 78, and
a vacuum source 80. The pressure and vacuum sources are in fluid
communication with a manifold 64, e.g. via tubing 84. The manifold
is also in fluid communication with the spotting capillaries so
that pressure or vacuum applied to the manifold is delivered to the
channel (bore) in the capillaries. In preferred embodiments, there
is a waste receptacle disposed between the low vacuum source 80 and
the manifold 74. In certain embodiments, vacuum source 80 supplies
various levels of vacuum.--a "high" vacuum for cleaning and for
starting the filling of the pins with a vacuum pulse, and a "low"
vacuum that is sometimes used to help capillary action complete the
filling of the pins. This low vacuum is typically low enough so
that the printing liquid is not pulled past the top of the printing
pins and into the flexible capillary tubing.
In preferred embodiments, the system is designed so that the tubing
from the waste bottle to the manifold preferably slopes downward to
facilitate liquid flow, and we strive to minimize the volume of the
tubing and waste receptacle so that the pressure changes are
transmitted to the manifold quickly. Thus, while the tubing
communicating the pressure and vacuum to the manifold and
communicating the manifold to the waste receptacle can be
essentially any convenient tubing (as long as it is resistant to
the reagents employed), in preferred embodiments, the tubing is a
low void volume tubing (e.g. a fine bore capillary tubing), but the
diameter is not so small as to introduce too much flow resistance.
Such tubings are well known to those of skill in the art.
In one preferred embodiment the pressure source 78 can apply two
pressures, a blowout pressure (e.g. 15 PSI), and a positive
pressure used during printing (e.g. from about 0.1 to 2, preferably
from about 0.1 to 1, more preferably from about 0.1 to about 0.5,
and most preferably about 0.3 inches of water). Similarly, in
preferred embodiments, the vacuum source 80, can apply two
pressures, a "high vacuum" for cleaning, and for starting filling
of the tubes using a short burst of vacuum, and a "low vacuum" to
assist capillary action in filling the printing pins. The waste
receptacle is preferably a low volume waste receptacle (e.g. a 200
ml waste bottle).
One suitable manifold is illustrated in FIG. 8. This manifold
comprises a common channel with an inlet port (manifold inlet) 66
and an outlet port (manifold outlet) 68. In fluid communication
with the manifold are a number of capillary connectors 76 that each
with an internal (manifold) capillary port 72, and an external
capillary port 68. Each capillary connector is disposed to receive
a connection (e.g. a tubing connection) to provide a fluid
communication to a spotting capillary 12. The internal capillary
port 72 is disposed inwards into the manifold so that the capillary
port is not flush with the internal wall of the manifold. This
prevents droplets from accumulating on the internal capillary port
72 which could interfere with reliable loading or delivery of
samples.
The manifold can be made of any of a variety of materials and can
take a number of different shapes. Preferred shapes however, permit
the rapid distribution of pressure, permit the unidirectional flow
of gas and waste, and permit the disposition of the internal ports
72 away from the internal surface of the manifold. Useful
materials, include various plastics, glass, quartz, ceramic, and
metals. In one preferred embodiment, the manifold is fabricated
from stainless steel.
A plumbed print head is illustrated in FIG. 9. This figure
illustrates the print head 10 comprising a plurality of spotting
capillaries 12 (four visible in the figure). The spotting
capillaries are in fluid communication with the manifold 64 via
flexible capillary tubing 70. A pressure line 84a can be seen at
the upper right and a waste/vacuum line 84b can be seen at the
upper left.
Another pressure/vacuum system is illustrated in FIGS. 10, 11 and
12. This pressure/vacuum system for managing the print head permits
operation in a larger format printer where tubing between the print
head and pressure and vacuum sources is sufficiently long so that
the volume and flow impedance of the tubing has a significant
impact on printer operation. The basic operating concept is to
assure that flow of gas or liquid in the manifold is always towards
the waste bottle as shown by the arrow in FIG. 10. This prevents
liquid that may be in present the line going from the manifold to
the waste bottle from flowing back into the manifold and blocking
the tubes that supply pressure and vacuum to the printing pins.
When large amounts of liquid are present in the manifold, for
example during a wash cycle, sufficient air flow is maintained in
the manifold to rapidly sweep the fluid into the waste bottle. The
controls for the pressure side of the system are illustrated in
FIG. 11, and those for the vacuum side are illustrated in FIG.
12.
As shown in FIG. 11, A pressure source 98 (e.g. N2 tank) is
connected through a printhead supply valve V1 100 to a medium
(blowout) pressure regulator R2 102 (e.g. .about.20 psi) and to a
very low (printing) pressure (e.g., .about.0-0.5 in H.sub.2O)
regulator R3 104. The regulators are optionally connected to a
blowout pressure readout 106, and/or to a very low pressure readout
108. The very low pressure side is connected through a very low
(printing) pressure valve V3 110 to a line 112 leading to the high
pressure side of the manifold 64. The medium pressure side is
connected through V3 medium (blowout) pressure valve V2 114 to a
line 90 leading to the high pressure side of the manifold 64. The
low pressure side is also vented to a vent through vent valve V0
116 to a vent 118.
As shown in FIG. 12 vacuum source 80 is connected to a coarse
vacuum regulator R4 132 maintaining a "vacuum" of about 20 in Hg,
and to a medium vacuum regulator R5 134 maintaining a "vacuum" of
about 15 in Hg. The two regulators can, optionally, be connected to
"coarse vacuum" and medium vacuum readouts 136 and 138,
respectively. The line from the coarse side vacuum regulator
connects via a coarse (wash) vacuum valve V4 140 to a waste
receptacle 82 which is also connected via a vacuum line 146 to the
manifold 64 as shown. The line from the coarse side vacuum
regulator connects via a vacum valve V5 142 to the waste receptacle
82 and via a vacuum vent valve VV 144 to a vent 72 as
illustrated.
Two features contribute to maintaining unidirectional flow and
rapidly clearing liquid from the manifold. Liquid clearing is
assured by the vent line 120 containing connecting to the vent 118
and containing a needle valve 122, which is controlled by the
"constrictor valve" 116 in FIG. 11. This has particular use when
washing the print head. At some times during the washing the tips
of the printing pins are immersed in liquid and a vacuum is
applied. This draws liquid into the pins and up into the manifold.
Opening constrictor valve 116, allows a small amount of air to
enter the pressure side of the system, and sweeps the liquid out of
the manifold and to the waste bottle. The amount of flow is
determined by adjusting the needle valve.
Unidirectional flow is accomplished always applying vacuum to one
side of the manifold, and pressure to the other, and by providing
atmospheric vents on both the pressure and vacuum sides of the
system. The vents are controlled by valves V0 116 and VV 144
respectively. In combination with valve CV 116, this permits
rapidly returning the system to atmospheric pressure without having
a period of flow in reverse direction.
For example, during a wash cycle where vacuum is applied, all of
the tubing in the system including that on the high pressure side
of the manifold, is below atmospheric pressure. If the only
atmospheric vent were on the vacuum side of the system, opening
that vent would result in flow from the low pressure side toward
the manifold as the tubing filled with air. Conversely, when the
system is pressurized in order to blow out any liquid that may be
in the printing pins, the tubing and waste bottle are at elevated
pressure. If the only atmospheric vent were on the high pressure
side of the system, then the gas in the waste bottle would flow
towards the manifold when this vent was opened. Both of these
reverse flows may be large enough to transport any liquid that may
be in the tubing connecting the manifold to the waste bottle back
towards the manifold, perhaps causing some to enter the manifold.
The flow reversals can either be entirely eliminated or reduced to
acceptable levels by using vents on both the pressure and vacuum
sides of the system. Opening both vent valves, V0 116 and VV 144,
simultaneously permits returning the system to atmospheric pressure
without substantial flow reversal. The dual vents V0 and VV also
provide the advantage of returning the system to atmospheric
pressure rapidly since VV can be physically close to the waste
bottle, reducing the flow impedance between it and the vent.
B) Representative Print Cycle.
Valve V1 100 is typically open continually during printing. A
typical print operation begins by cleaning the printing pins. The
tips of the pins are dipped into a sonication bath containing a
dilute solution (approximately 1 part in 10,000 in distilled water)
of glass cleaning solution, (Micro 90). Pressure may be applied to
expel the contents of the pins by opening valve V2 114 or V3 110.
Subsequently, vacuum is applied to the manifold by opening valve
V4. Valve CV is also opened to provide air flow through the
manifold. The vacuum draws cleaning fluid into the printing pins
and up to the manifold, where the air flow sweeps it rapidly to the
waste bottle. The print head is dipped into the cleaning fluid
several times with the vacuum applied so that some air enters the
printing pins when they are raised above the fluid. Each dip cycle
last approximately 1 sec. Alternating regions of air and liquid
travel up the printing pins and their associated tubing to the
manifold. The interspersed regions of liquid and air provide more
rapid removal of residual printing solutions from the printing pins
than would be obtained if the tips of the pins were just dipped
into the wash solution and remained continuously immersed.
The print head is then moved over a second sonication bath that
contains distilled water and the tips of the pins are periodically
immersed several times with valves V4 140 and CV 116 open. Each dip
cycle last approximately 1 sec. This draws water through the pins
to remove the cleaning solution. The print head is then moved to a
drying station where warm air is blown over the printing pins for
approximately 5-10 seconds. During this time, pressure may be
applied for a period to blow liquid out of the pins by opening V2
114 and closing the other valves. The remainder of the drying time
vacuum is applied by opening V4 140, and CV 116 is opened to allow
airflow to dry the manifold.
After the drying period V4 and CV are closed and atmospheric vents
VV 144 and V0 116 are opened to bring the system to ambient
pressure. The print head is then positioned over the microtiter
plate and the tips of the printing pins are immersed in printing
solutions from the desired position in the plate. VV 144 and V0 116
are closed. A vacuum pulse is applied by opening V5 142 in order to
begin the loading of printing solutions into the pins. After
.about.0.1-. 1.0 sec V5 is closed and the atmospheric vents VV 144
and V0 116 are opened to return the system to ambient pressure.
Loading of the printing solutions continues for .about.1-2 sec
using capillary action. The printing pins are then lifted out of
the printing solutions and VV and V0 closed.
The print head is then moved to its printing position and valve V3
110 is opened to apply a low, constant, pressure of 0.0-0.5 inches
of water to the printing pins. This pressure assures that the
printing solutions remain at the tips of the printing pins so that
the tips remain wet with printing solution. Varying the pressure
provides some control over the amount of liquid that is deposited
when the printing pins contact the array substrates. The pressure
is kept low enough so that the printing solutions are not ejected
from the pins. The tips of the pins are contacted with the array
substrates at all of the locations specified by the operator. After
completion of the printing with the current load of printing
solutions, the cycle starts again with the print head being moved
over the first sonicator bath, the tips lowered into it, and the
unused printing solutions expelled.
V. Preparation of a Microarray
The microarray printer of this invention can be used to print
microarrays comprising essentially any molecules that can be
suspended, dissolved, or otherwise placed in a solution. Preferred
microarrays include, but are not limited to microarrays of
biomolecules (e.g. sugars, carbohydrates, nucleic acid, proteins,
and the like). Particularly preferred microarrays include nucleic
acid and/or protein arrays. Methods of preparing and/or purifying
biomolecules are well known to those of skill in the art (see,
e.g., Berger and Kimmel (1989) Guide to Molecular Cloning
Techniques, Methods in Enzymology 152 Academic Press, Inc., San
Diego, Calif.; Sambrook et al. (1989) Molecular Cloning-A
Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor Press, NY; Ausubel et al. (1994)
Current Protocols in Molecular Biology, Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (1994 Supplement) (Ausubel); U.S. Pat. No.
5,017,478; and European Patent No. 0,246,864.).
The materials that are to be printed (e.g. proteins, nucleic acids,
etc.) are typically formulated in "printing solutions". Solutions
for microarray printing are well known to those of skill in the art
(see, e.g., U.S. Pat. Nos. 6,101,946, 5,958,342; and MacBeath and
Schreiber (2000) Science 289: 1760-1763; Mark Schena (Ed.) (1999)
Genes, Genomes and Chips. In DNA Microarrays: A Practical Approach,
Oxford University Press, Oxford).
The microarrays can be fabricated on any of a wide variety of
substrates well known to those of skill in the art. Such substrates
include, but are not limited to glass, plastic, quartz and other
minerals, metal, ceramic, porcelain, metal covered (e.g. sputtered)
glass, and the like. A number of substrates, often derivatized to
facilitate microarray printing are commercially available (see,
e.g., silane slides from Sigma Chemical Co., the SuperClean.TM.,
SuperAmide.TM., and SuperAldehyde.TM. substrates from Telechem,
International Inc., etc.).
In operation, the printing pins (spotting capillaries) are
initially washed. In a preferred embodiment, this involves moving
the print head over a wash bath and applying pressure (e.g., about
15 PSI) to the manifold to blow out any liquid remaining in the
spotting capillaries. The spotting capillaries are dipped in and
out of a cleaning solution (e.g. 0.001% Micro-90, Cole Palmer
Inc.), in a sonicating bath at about 0.75 sec intervals for about 3
cycles while pressurized.
The device is switched from pressure to house ("high") vacuum and
the spotting capillaries are dipped into the sonicated cleaning
solution for 3 more cycles while drawing cleaning solution into the
spotting capillaries. The spotting capillaries are then moved to a
sonicating rinse bath that contains pure (e.g. double distilled)
water.
The spotting capillaries are dipped in and out of water for about 3
cycles of about 0.75 sec each, with vacuum applied. This allows the
formation of interspersed air bubbles and water in the tubing,
assuring that the cleaning solution is more effectively removed
from the walls of the tubes. Finally the spotting capillaries are
dried by sucking air through them using house vacuum, blowing hot
air over the spotting capillaries for about 4 sec, opening a vent
to atmosphere and continuing vacuum for about 2 sec in order to
clear liquid from the manifold and waste tubing (see FIG. 7).
The spotting capillaries are then filled by dipping the spotting
capillaries into reagent reservoir(s) (e.g. a microtiter plate)
containing the printing solution(s). Full house vacuum is applied
to the manifold for about 0.15 sec in order to assure that the
solutions enter the tips of the pins. The manifold is then vented
to atmospheric pressure and the spotting capillaries are allowed to
sit in the printing solutions for about an additional 0.75 sec so
that capillary action fills the spotting capillaries. In one
preferred embodiment, the spotting capillaries each contain
approximately 0.2 ml when full. The spotting capillaries do not
fill beyond their tops due to capillary action--the solutions do
not enter the flexible tubing that connects the pins to the
manifold. In some cases a slight vacuum of .about.0.2 inches of
water is used to assist the filling. This is adjusted to be low
enough so that the no liquid is drawn beyond the top of the pins.
The entire wash, dry and fill functions take about 25 seconds.
To print an array feature or features, the print head is moved over
the first printing substrate and lowered to make contact, and
raised. In one preferred embodiment, in the upper position the tips
of the printing pins are about 0.5 to 1.0 mm above the array
substrate, and when printing the print head is lowered so that the
pins would move about 0.2 mm below the substrate surface if no
array substrate were present. When a substrate is present the
spotting capillary tips contact it and the spring mounts allow the
spotting capillaries to stop moving while the print head body
continues its motion toward the substrate. In a preferred
embodiment, the total time for the print head to move down, contact
the slide and return to the upper position is about 0.05 to 0.2
sec. During the printing operation a constant pressure of 0.1 to
0.4 inches of water is applied to the manifold to keep the printing
solutions at the tips of the spotting capillaries. This assures
that the spotting capillaries are wet with the printing solutions
so that liquid will be transferred to the substrate on contact. The
pressure does not eject the printing solution. If this is not done,
the solutions can pull away from the tips and the printing will
stop. The small diameter of the tubing and the printing pins
provides enough flow resistance to air so that the manifold
pressure is maintained even if one or more of the pins does not
contain printing solution. The cycle is repeated to print
additional features or other array substrates.
The amount of printing solution that is deposited on the substrate
depends on the interaction between the substrate and the printing
solution, and the diameter of the tip of the printing pin. When
printing salt solutions on glass, each fill of the printing pin
(0.2 ml) can make at least 10,000 spots. When printing 20% DMSO
solutions with DNA, the same load can print at least 2000 spots.
This is many more spots than are possible with other printing
systems. Thus the system described above can deposit less than 100
pL per spot in typical printing.
Use of the novel spotting capillaries and/or print heads of this
invention provides extremely efficient reagent usage. In certain
embodiments, the printer can print at least about 500 spots
(features) per 0.2 .mu.L load, more preferably at least about 1000
spots (features) per 0.2 .mu.L load, most preferably at least about
1500 spots (features) per 0.2 .mu.L load, at least about 2000 spots
(features) per 0.2 .mu.L load, at least about 5,000 spots
(features) per 0.2 .mu.L load, or at lest about 10,000 spots
(features) per 0.2 .mu.L load. Because the printing capacities are
so high, the print head typically does not need to refill during a
print run. This greatly decreases the duration of a print run when
a large number of arrays are made at one time.
VI. Microarray Printing Device
The various elements described above, e.g. spotting capillaries,
print head design, array support platen, print head support platen,
vacuum and pressure system, manifold, sample loading unloading
protocols, and the like can be incorporated individually, or in
combination, into preexisting microarray printers or they can be
assembled into a microarray printer built de novo.
Methods of designing and building microarray printing devices are
generally known to those of skill in the art (see, e.g., U.S. Pat.
Nos. 6,110,426, and 5,807,522, and publications of the Brown
Laboratory at Stanford University (e.g., The McGuide. Version 2.0,
available on the internet at
http://cmgm.stanford.edu/pbrown/mguide/index.html, and from Cold
Spring Harbor Laboratories).
In general, microarray printers of this invention will, in
preferred embodiments, include a base adapted to hold reservoir(s)
of printing solutions, a platen for supporting and positioning
microarray substrates, and a platen for supporting and positioning
a print head. The microarray printer will include actuators (e.g.
motors) for driving/positioning the various platens and for
vertically positioning the print head. The microarray printer, will
typically include associated electronics to read encoded platen
positions and/or to drive the various actuators to position the
array substrates and print head. Typically such electronics will
include a computer controller. The microarray printer can
additionally comprise vacuum and pressure lines, reagent
reservoirs, waste receptacles, cleaning baths and the like as
described herein.
In a particularly preferred embodiment, the microarray printer will
include the print head platen, the array substrate platen, a print
head comprising spotting capillaries as described herein. The
microarray printer will also preferably also include pressure and
vacuum sources as described herein. While it is preferred that the
microarray printer comprise all of the elements described herein,
it is not required that all such elements be present. Thus, in
certain embodiments, the microarray printer comprises one, two, or
only a few of such elements. Thus, for example certain microarray
printers may only comprise a print head according to this invention
and/or the array substrate platen, and/or the print head support
platen, and so forth.
VII. Microarrays
It is believed that the microarray printers of this invention
permit the production of spotted microarrays with an accuracy,
consistency, and array feature density previously unavailable.
Thus, in certain embodiments, this invention provides high-density
microarrays comprising a plurality of molecules, preferably
biomolecules where the array comprises at least about 1,000
features (spots), preferably at least about 10,000 features
(spots), more preferably at least about 40,000 features (spots),
and most preferably at least about 100,000 features (spots), or at
least about 1,000,000 features (spots). In particularly preferred
embodiments, the features are present at an average
center-to-center spacing of about 130 .mu.m or less, preferably
about 100 .mu.m or less, more preferably about 80 .mu.m or less,
and most preferably about 65, 50, or 40 .mu.m or less.
In certain embodiments, the microarray is a protein and/or nucleic
acid microarray. In nucleic acid arrays unlike chemically
synthesized arrays, the printed arrays of this invention are not
limited by the size of nucleic acid. Large nucleic acids can be
printed as easily as small nucleic acids (e.g. oligonucleotides
less than 20-30 mer). Indeed, there is no size limit on the printed
nucleic acids and the particular nucleic acid sizes depends on the
intended use of the array. Arrays printed according to the methods
of this invention typically have a fragment size ranging from about
100 to about 1000 bases (e.g. a mixture of PCR fragments). Thus,
frequently nucleic 100 nucleotides or longer are printed. Fragment
sizes ranging from about 1000 bases to about 10,000 bases or from
about 10,000 bases to about 100,000 bases or larger can be readily
accommodated.
Preferred arrays of this invention have a feature (spot) density
greater than about 5,000, 10,000, or 20,000 features/cm.sup.2,
preferably greater than about 30,000 features/cm.sup.2, more
preferably greater than about 40,000 features/cm.sup.2, and most
preferably greater than about greater than about 50,000 or 60,000
features/cm.sup.2.
Because the the reagents are typically simply spotted, in preferred
embodiments, the molecule(s) comprising the array features are
simply adsorbed to said substrate. However, in certain embodiments
the reagents and/or the array substrate can be derivatized so that
the molecules comprising the features (spots) covalently couple to
the substrate. Methods of so derivatizing macromolecules are well
known to those of skill in the art. Thus, for example, the reagents
can be derivatized with a sulfhydryl group (--SH) which will
covalently couple to a gold surface (e.g. gold coated glass).
VIII. Kits
In still another embodiment, this invention provides kits
comprising one or more containers containing the arrays described
above. In certain embodiments, the arrays will comprise features
representing nucleic acids from every chromosome in a subject
organism (e.g. a human). In certain embodiments, the arrays will
comprise features representing nucleic acids from every known
expressed sequence tag (EST) for a given organism, or tissue, or
whose expression is associated with a particular physiological
state (e.g. a particular pathology).
These array constituents are merely illustrative. Numerous other
arrays components will be recognized by one of ordinary skill in
the art.
In certain embodiment, the kits can, optionally, additionally
contain one or more of the following: detectable labels,
hybridization reagents, software, buffers, and the like.
* * * * *
References